SUMMARY Retinal rods and cones signal the presence of light by hydrolyzing cGMP which then closes cyclic nucleotide gated cation channels in the plasma membrane. An inward Na+ current is interrupted and the cell hyperpolarizes. It has been shown that bicarbonate can increase the flash response amplitude and quicken response kinetics. However, many features of this modulation are not understood including: the full magnitudes of the effects, the dose-response relations, the intra-retinal sources of bicarbonate for photoreceptors, the pathway(s) for access of bicarbonate to the outer segment, differences between rods and cones, and the mechanism of bicarbonate action. Using single cell recording and biochemical assays, we will tackle each of these issues. Rods have an anion exchanger in their outer segments that normally extrudes bicarbonate. But by removing extracellular Cl-, we can force the anion exchanger in rod outer segments to take up bicarbonate. We will then describe how bicarbonate affects the amplitude and kinetics of both bright flash and dim flash responses in single cell recordings of membrane current. The bright, saturating flash response provides a measure of the circulating current. Responses kinetics vary with flash strength but become invariant with very low flash strengths, so the dim flash response is useful for analysis of response kinetics. To find out how bicarbonate normally accesses the rod outer segment when extracellular Cl- is present, we will change the recording configuration: outer segment inside the pipette vs inner segment inside the pipette, isolated rod vs rods attached to a piece of retina. We will explore endogenous bicarbonate modulation of phototransduction in cones by applying a carbonic anhydrase inhibitor while recording from isolated cones and cones attached to pieces of retina and begin to elucidate conditions in which dynamic modulation comes into play. To fully characterize the effects of bicarbonate on cones for comparison to results from rods, we will carry out experiments with exogenous bicarbonate, similar to those proposed for rods except that a carbonic anhydrase will be present. In elucidating the mechanism of bicarbonate, we will consider three targets: guanylate cyclase, PDE, cyclic nucleotide gated channel. The first two targets will be evaluated in biochemical assays, while the third will be tested in excised membrane patch recordings. Some evidence already suggests that bicarbonate stimulates ROS- GC1 activity. It then becomes important to find out whether ROS-GC2 is similarly affected, to define the bicarbonate binding site of each ROS-GC, and to determine the calcium dependence of bicarbonate stimulation when the calcium sensing subunits GCAPs and S100B are bound to ROS-GCs. A long term goal is to dissect the molecular mechanisms that shape the photon response under light and dark adapted conditions and to understand their roles, be it causative or modulatory, in retinal disease.